By increase of the population of obese people and life expectancy caused by fast growing industrialization, the diabetes mellitus (DM) is easy to find in the people of all ages, and the number of such people continues to increase.
What is most frequently used as a key index reflecting the level of blood glucose in the diabetes mellitus (DM) is blood glucose and glycated hemoglobin (HbA1c). Since the glycated hemoglobin is known to cause less errors in examination conducted based on empty stomach and taking medicine. With the aid of the glycated hemoglobin, it is possible to estimate a long term development of the previous level of blood glucose before the moment blood was collected from a diabetic patient, for thereby usefully controlling and evaluating blood plasma for a long time period. Since the specificity is good, the glycated hemoglobin may be a good index for estimating diabetes and its complication. In consideration that the lifespan of red blood cell is 120 days, the glycated hemoglobin well reflects the average state of blood glucose for the recent 1˜3 months, more particularly, the glycated hemoglobin intensively reflects a change in the blood glucose for one month period.
In addition to the blood glucose, the glycated hemoglobin is regarded as an important index for judging the diagnosis and treatment of diabetes through the examination of glycated hemoglobin. In the year of 2010, the American Diabetes Association (ADA) more actively updates the diagnosis standards which recommend performing the glycated hemoglobin examination during the diagnosis of diabetes.
The fixed criteria of the glycated hemoglobin may be expressed in a form of the percentage (%) of fraction of the glycated hemoglobin with respect to the total quantity of hemoglobin. In case of a normal person who is not a diabetic patient, they have glycated hemoglobin of below 5˜6%. In case of a diabetic patient, the ratio of the glycated hemoglobin in blood is in excessive of 7%, and in case of a person who has a glycated hemoglobin ratio of around 7%, a more aggressive control and measurement with respect to the level of blood glucose is necessary through an additional management. Therefore, the detected concentration which has the most important meaning in terms of the measurement of glycated hemoglobin is a 7% section of glycated hemoglobin. For this reason, the development of a glycated hemoglobin measuring system which is capable of precisely differentiating the levels between 6.9% and 7% based on the above 7% section is required.
The development and application of the reactive layer which has a high selectivity and reactive efficiency with respect to the detection target substances are important so as to precisely and sensitively detect the target substances. Here, the reactive layer should selectively react with respect to a small quantity of detection target substance, and a high level of reaction yield should be guaranteed. For this, a recognition substance reacting with the detection target substance should exist in the reactive layer with a high concentration.
Generally, for immobilization of the target recognition substance with respect to a bio-sensing substrate, methods such as a simple adsorption method, a static electricity-based binding method, a self-assembled monolayer (SAM), etc. have been used. Especially, with the SAM technology, a well arranged 2D biological substance may be fixed on the surface of a solid substrate. The SAM technology is widely used in manufacturing an electrochemical and optic biosensor, since the SAM technology provides a relatively easier surface modification and biological substance treatment process as compared with other technologies.
However, the detection target substance can bind only with a monolayer structure over a limited 2D plane area on the reactive layer to which the SAM technology is applied. Thus, this may cause a spatial limitation to the binding reaction with more detection target substances. Also, it is disadvantageous that the SAM technology may be applied only to the limited materials such as a gold- or chemical-processed silicon substrate.
To resolve the above problems, a big attention is paid on developing and introducing a new structured reactive layer having a 3D structure rather than a 2D structure, wherein a target recognition substance is integrated with a high density in a corresponding structure to more efficiently and sensitively recognize a detection target substance. In addition, the development of a reactive layer which may be applied to a substrate made from a more economical material like a paper or a membrane, not a substrate which has a high manufacturing cost like the typical metallic electrode, etc. is required.
As for the glycated hemoglobin biosensor, it is reported that a boronic acid (BA), which can binds with a saccharide substance via cis-diol bond, is used as a recognition substance for glycated hemoglobin, and a boronic acid SAM may be formed at the surface of a gold electrode using a substance like a mercaptophenyl boronic acid. However, such methods may have limits in the glycated hemoglobin binding yield due to the 2D space limitation, and in that they are only applicable on the metal surface.
Therefore, in order to implement more sensitive glycated hemoglobin detection, the development of a new structured reactive layer wherein a boronic acid is arranged with a high density in a 3D structure in the limited area is required.